激素非依赖性前列腺癌的治疗进展

前列腺癌是欧美国家男性患者中最常见的肿瘤.在美国前列腺癌影响着1/6男性,超过200万人患有此病.在我国前列腺癌(PC)的发病率也在不断增加.该疾病如果早期发现,癌细胞局限在前列腺内,外科手术非常有效,几乎100%患者可以在几年之内保持无癌状态[1].然而,癌细胞一旦扩散,其后果几乎是致命的.     

向日葵盘黄酮对前列腺癌DU145细胞的抑制作用

目的探讨向日葵盘黄酮对前列腺癌细胞株DU145的影响。方法应用不同浓度向日葵盘黄酮培养DU145细胞,倒置显微镜观察细胞形态,检测细胞的生长抑制率、细胞凋亡情况及细胞周期。结果不同浓度的黄酮化合物均对细胞生长具有明显的抑制作用,且随着浓度的增加和作用时间的延长,对DU145细胞的生长抑制率逐步提高。倒置显微镜显示细胞有凋亡小体等形成,且琼脂糖凝胶电泳显示不同浓度黄酮作用DU145细胞24h后,细胞发生了凋亡,并且处在晚期凋亡阶段。流式细胞仪检测显示不同浓度黄酮作用细胞48h后,随着黄酮浓度的增高,DU145细胞S期细胞增多,M期细胞有减少趋势,同时可检测到凋亡细胞的亚G1峰。结论向日葵盘黄酮可抑制人前列腺癌细胞株DU145的生长,能使细胞凋亡增加,G/M期细胞百分数升高,并能引起细胞凋亡相关基表达改变。     

全雄激素阻断治疗对老年非转移性前列腺癌患者认知功能的影响

目的探讨全雄激素阻断治疗(CAB)对老年非转移性前列腺癌患者认知功能的影响。方法根据患者是否施行根治性前列腺切除术将79例老年非转移性前列腺癌患者分为CAB组40例(未行前列腺切除术)和对照组39例(行前列腺切除术)。采用蒙特利尔认知评估量表(MoCA量表)评价两组治疗前、治疗6、12个月后的认知功能。结果治疗6、12个月后,CAB组Mo CA总分、记忆、视空间与执行功能、注意得分以及血睾酮水平均显著低于对照组(P0.05)。CAB组治疗6、12个月后的血睾酮水平均与同期Mo CA总分呈正相关(r=0.235、0.221,P0.05)。结论CAB可显著损害老年非转移性前列腺癌患者的认知功能,早期主要以记忆、视空间与执行功能、注意功能受损为主。     

雄激素受体对前列腺癌PC3细胞株增殖能力的影响

目的研究雄激素受体（AR）对雄激素非依赖性前列腺癌细胞株PC3增殖能力的影响。方法将含有AR的质粒（Pbabe-AR）稳定转染到PC3细胞,建立起新的细胞系PC3-AR,并用实时逆转录聚合酶链反应（Q—PCR）、蛋白免疫印迹试验（Western blot）和荧光素酶法确认AR的表达水平和功能;用MTT法、软琼脂糖凝胶实验检测AR对体外培养PC3细胞增殖的影响。结果PC3-AR细胞能够稳定表达有功能的AR;MTT实验显示PC3-AR细胞体外生长慢于PC3-v细胞,加入DHT以后效果更加明显。相对于PC3-v,PC3-AR在软琼脂糖凝胶中形成的克隆明显减少。结论AR能够降低PC3-AR细胞在体外的增殖能力。     

关于激素非依赖性前列腺癌的几点思考

激素非依赖性前列腺癌是前列腺癌治疗中的重点和难点之一。总的来说前列腺癌从激素依赖向激素非依赖发展的机制尚不完全清楚,许多难点尚未破解,临床疗效尚不满意。近年来,欧美各国对此投入相当大的人力物力财力进行研究,获得了一些突破,取得了一一定共识：过去山于患者较少,国内对激素非依赖性前列腺癌的诊治手段匮乏。     

激素难治性前列腺癌治疗进展

前列腺癌是男性泌尿生殖系统常见肿瘤之一，在美国占男性肿瘤死亡率第二位，近年来在我国的发病率和死亡率也逐年升高。前列腺癌是雄激素依赖性肿瘤，所以大多数前列腺癌开始对祛除雄激素治疗是有效的，但大多数患者在接受内分泌治疗后18-24个月会转变成为激素难治性前列腺癌（hormone refractory prostate cancer．HRPC），而HRPC的治疗一直是前列腺癌治疗的难点。     

EGF与miRNA-21联合构建雄激素非依赖性前列腺癌LNCaP亚细胞模型

目的构建雄激素非依赖性的人前列腺癌细胞株LNCaP亚型。方法前期常规RPMI 1640培养基培养LNCaP细胞,然后给予无酚红的RPMI 1640培养基,其中加入10%活性碳/葡聚糖处理的胎牛血清,细胞传代后将10μg/L表皮生长因子（EGF）加入培养基,50 nmol/L miRNA-21类似物转染细胞,长期培养。镜下观察细胞形态变化,MTT检测细胞增殖活性,RT-PCR检测雄激素受体基因扩增情况。结果细胞培养约2.5个月后,LNCaP细胞成功转变为非雄激素依赖的前列腺癌细胞LNCaP亚细胞,细胞增殖能力升高,雄激素受体表达上调。结论体内雄激素依赖性前列腺癌可向雄激素非依赖性前列腺癌转变,而且miRNA-21和EGF对该转变起促进作用。     

SIRT1抑制剂sirtinol对前列腺癌DU145细胞凋亡的影响及可能机制

目的观察SIRT1抑制剂sirtinol对前列腺癌DU145细胞系细胞凋亡和细胞凋亡关键调控因子(Bcl-2、Bax、Cytochrome C和Caspase-3)表达变化的影响,探讨SIRT1在前列腺癌发生的可能机制。方法体外培养DU145细胞,实验分对照组(DMSO组)和sirtinol组(终浓度为10,25和50μmol/L),Western印迹检测DU145细胞SIRT1蛋白表达水平;流式细胞术检测细胞凋亡;Western印迹检测DU145细胞中细胞凋亡关键调控因子Bcl-2、Bax、Cytochrome C和Caspase-3的蛋白表达。结果与对照组相比,随着sirtinol浓度的增加,SIRT1蛋白表达水平逐渐降低,细胞凋亡比例增加,诱导细胞发生凋亡。不同浓度sirtinol作用DU145细胞后Bcl-2蛋白表达减少,且随剂量增加表达越低;Bax蛋白表达增加,Bcl-2/Bax比率降低;伴随Cytochrome C的释放和Caspase-3的激活。结论下调SIRT1表达诱导前列腺癌细胞DU145细胞凋亡,其机制可能与改变细胞凋亡关键调控因子Bcl-2、Bax、Cytochrome C和Caspase-3的蛋白表达相关。     

DHT-PROTACS对前列腺癌细胞分泌PSA的抑制情况观察

雄激素依赖性前列腺癌细胞(LNCaP细胞)和雄激素非依赖性前列腺癌细胞(LNCaP-C81细胞)分别培养,各自分为对照组和实验组.两个实验组均给予双氢睾酮(DHT)-PROTACS,4组细胞培养后分别取培养液上清并检测PSA表达的情况.LNCaP-C81对照组、实验组和LNCaP对照组、实验组的PSA分泌值分别为(411.89±22.25)、(102.66±12.06)、(339.81±26.41)和(123.85±16.84)ng/ml.各实验组和对照组比较,P均＜0.05.认为DHT-PROTACS处理前列腺癌细胞(LNCaP细胞和LNCaP-C81细胞)后,均能导致前列腺癌细胞分泌PSA减少.     

多烯紫杉醇联合AG490对前列腺癌DU-145细胞增殖凋亡的影响

目的 探讨多烯紫杉醇联合酪氨酸激酶(JAK)抑制剂AG490对前列腺癌DU-145细胞增殖凋亡的影响,并探讨其机制.方法 将培养后的对数生长期前列腺癌DU-145细胞随机分为紫杉醇组、AG490组、联合组及对照组.紫杉醇组、AG490组、联合组分别采用不同浓度的多烯紫杉醇、AG490及二者联合应用干预;对照组不干预.干预24、48 h后采用MTF法检测各组细胞生长抑制率;流式细胞仪检测细胞凋亡率;干预48 h采用免疫组化法检测各组Bcl-2表达.结果 紫杉醇、AG490及联合组细胞生长抑制率均明显高于对照组,呈时间和剂量依赖性;联合组明显高于紫杉醇组及AG490组,P均＜0.05.紫杉醇组与AG490组比较无统计学差异.各组细胞凋亡率比较亦呈现相同趋势.各干预组细胞Bcl-2蛋白表达均明显低于对照组,联合组Bcl-2蛋白表达明显低于紫杉醇组与AG490组(P均＜0.05).结论 多烯紫杉醇与AG490均可抑制DU-145细胞增殖,诱导其凋亡,呈剂量、时间依赖性;二者联合应用有协同作用,其作用可能与Bcl-2介导有关.     

Celecoxib对胃癌细胞凋亡的影响

目的:研究选择性环氧合酶-2(COX-2)抑制剂Celecoxib对人胃癌细胞株SGC7901凋亡的影响.方法:采用MTT法测定人胃癌细胞株SGC7901分别在1×10-5,1×10-6,1×10-7,1×10-8,1×10-9mol/L的Celecoxib作用48h后生长抑制情况;流式细胞术(FCM)观察Celecoxib对SGC7901细胞凋亡的影响;采用免疫细胞化学染色观察Survivin的表达.结果:Celecoxib浓度为1×10-5-1×10-9mol/L时,对SGC7901细胞的生长均有抑制作用,以1×10-5mol/L浓度的抑制作用最为显著,其抑制率为30.03%;1×10-5mol/LCelecoxib作用12,24,48h后,细胞凋亡比例较对照组均明显增加,48h凋亡率达17.83%;1×10-5mol/LCelecoxib作用后,Survivin的表达自用药3h起下降,24h最明显.结论:Celecoxib可诱导SGC7901细胞的凋亡,其可能的作用机制为抑制细胞Survivin基因的表达.     

吲哚美辛对前列腺癌DU145细胞增殖与凋亡的影响及机制探讨

目的观察吲哚美辛对人激素非依赖性前腺癌细胞系DU145增殖和凋亡的影响,并探讨其作用机制。方法采用不同浓度的吲哚美辛处理DU145细胞。应用四甲基偶氮唑蓝（MTT）比色法检测并计算细胞增殖抑制率;流式细胞术检测细胞周期分布,并计算细胞凋亡率;采用酶联免疫吸附测定（ELISA）法测定细胞培养上清PGE2;应用RT-PCR法检测Bcl-2和Bax mRNA。结果吲哚美辛可明显抑制DU145细胞增殖（P〈0．05）,并具有时间一剂量依赖性;经吲哚美辛作用后,S期细胞明显增多,G2/M期细胞减少,细胞出现凋亡峰;细胞上清中PGB量明显减少（P〈0．001）;Bcl-2 mRNA表达降低,BaxmRNA表达下降,Bcl-2／Bax下降（P〈0．05）。结论吲哚美辛对前列腺癌细胞DU145生长具有抑制作用,阻滞细胞向c2／M期转化,并促进其凋亡。其作用可能与抑制细胞PGE2释放、下调Bel-2／Bax有关。     

GABA对胰腺癌细胞株SW1990生长的影响

目的:观察γ-氨基丁酸(GABA)对胰腺癌SW1990细胞生长的影响．方法:采用MTT法和流式细胞术检测GABA对胰腺癌SW1990细胞增殖、细胞凋亡、细胞周期的影响,放射免疫分析法检测cAMP含量．结果:不同浓度的GABA(20-320μmol／L)促进胰腺癌SW1990细胞的生长,促进细胞周期比例的分布,使G0／G1细胞减少,S期和G2／M细胞增多,同时促进胰腺癌SW1990细胞内cAMP含量的增加,且呈剂量依赖性(P<0．01)．显著抑制细胞凋亡,凋亡率由27．4％降低到5．4％(X2=10．19,P<0．01)．结论:GABA促进胰腺癌SW1990细胞生长,可能通过受体后信息介导．     

脂联素对结肠癌HT-29细胞的增殖及凋亡研究

目的探讨脂联素对结肠癌HT-29细胞的增殖、凋亡的影响。方法以不同浓度脂联素干预细胞,MTT法检测细胞增殖能力;AnnexinV／PI双染后流式细胞仪检测细胞凋亡。结果随着脂联素浓度的不断升高和培养时间的延长,HT-29细胞的生长逐渐受到抑制,脂联素可以诱导细胞凋亡。结论脂联素可抑制HT-29细胞增殖,诱导细胞凋亡。     

Cell-cycle arrest and p53-independent induction of apoptosis in vitro by the new anticancer drugs 5-FdUrd-P-FdCydOct and dCydPam-P-FdUrd in DU-145 human prostate cancer cells

 Purpose: Current therapies have limited impact on the progression of metastatic hormone-refractory prostate cancer. Therefore, we investigated the utility of new heterodinucleoside phosphate dimers of 5-fluorodeoxyuridine (5-FdUrd) in p53-mutated and androgen-independent DU-145 human prostate tumour cells. Methods: The effects of the dimers were assessed in vitro by a cell proliferation assay for cytotoxicity, flow cytometry for cell cycle distribution, confocal laser scanning microscopy for the detection of apoptotic bodies, poly(ADP-ribose) polymerase cleavage for caspase 3 activity and by a thymidylate synthetase assay. Results: The new dimers N ⁴-palmitoyl-2′-deoxycytidylyl-(3′→5′)-5-fluoro-2′-deoxyuridine (dCydPam-P-FdUrd) and 2′-deoxy-5-fluorouridylyl-(3′→5′)-2′-deoxy-5-fluoro-N ⁴-octadecylcytidine (5-FdUrd-P-FdCydOct) caused marked cytotoxicity with IC₅₀ values of 3–4 μM. 5-FdUrd-P-FdCydOct at 200 μM was capable of eradicating 100% of tumour cells whereas 10% of the cells were resistant to 5-FdUrd. Cytotoxicity was caused by a dramatic S-phase arrest, resulting in an increase of this cell population from 34% to 85% with 5-FdUrd-P-FdCydOct and to 81% with dCydPam-P-FdUrd. S-phase arrest was followed by apoptosis, as shown by 85% of the cells staining positive for Apo 2.7 antibody, a six- to eight-fold increased caspase 3 activity and DNA fragmentation. Thymidylate synthase activity was inhibited by 50% at 0.6–0.7 μM dimer concentration. The dimers were hydrolysed in vitro by phosphodiesterase I and human serum to the corresponding nucleosides and nucleoside monophosphates. Conclusions: The new dimers dCydPam-P-FdUrd and 5-FdUrd-P-FdCydOct are effective prodrugs of 5-FdUrd and have potential value for the treatment of p53-mutated and hormone-independent human prostate carcinomas. Received: 20 August 1999 / Accepted: 4 December 1999     

Effect of p27mt gene on apoptosis of the colorectal cancer cell line Lovo

AIM： To construct p27mt recombinant adenovirus, transfect the colorectal cell line Lovo and observe the effects of p27mt on Lovo cell apoptosis and cell cycle inhibition. METHODS： We constructed recombinant adenovirus containing p27mt by homologous recombination in bacteria. The colorectal cancer cell line Lovo was infected with recombinant replication-defective adenovirus Ad- p27mt, and expression of p27mt was determined by Western blotting; the inhibitory effect of p27mt on Lovo cells was detected by cytometry. Cell cycle was determined by flow cytometry. DNA fragment analysis identified the occurrence of apoptosis. RESULTS： The recombinant adenovirus which already contained p27mt target gene was successfully constructed. When multiplicity of infection was ≥50, the infection efficiency was 100%. After transfection of Lovo cells with Ad-p27mt the cells had high p27 expression which was identified by immunoblotting assay. PI staining and flow cytometry showed that 77.96% of colorectal cancer cells were inhibited in phase G0/G1, while in the Ad-LacZ group and blank control group, 27.57% and 25.29% cells were inhibited in the same phase, respectively. DNA fragment analysis, flow cytometry and TUNEL assay demonstrated that p27mt is able to induce apoptosis in colorectal cancer cells. CONCLUSION： p27mt has an obvious blocking effect on colorectal cancer cell cycle, and most cells were inhibited in phase G0/G1. Therefore, p27mt can induce apoptosis in colorectal cells.     

体外培养前列腺癌DU145细胞侵袭和迁移能力与EF-1 alpha基因表达的关系

目的 应用RNA干扰技术研究EF-1 alpha基因在体外培养前列腺癌细胞株DU145的侵袭和迁移中的作用. 方法将DU145细胞系分为3组:对照组(未转染siRNA),转染对照组(转染siRNA)和实验组(转染EF-1 alpha-siRNA).通过免疫荧光技术验证RNA干扰结果,并研究EF-1 alpha在DU145细胞中的定位以及与F-actin的定位关系.应用Transwell实验,比较EF-1alpha蛋白质水平调节前后各组肿瘤细胞侵袭和迁移能力的变化. 结果应用RNA干扰技术可以特异高效调低DU145细胞中EF-1 alpha蛋白质水平.EF-1 alpha蛋白质定位于细胞胞浆内,且该蛋白与F-actin存在共定位关系.但EF-1 alpha表达的改变不影响F-actin的分布.细胞迁移和侵袭能力研究结果显示,20×104个DU145细胞种入Transwall小室12 h后,对照组,转染对照组和实验组穿膜细胞数日分别为(10.6±1.0)×104个、(11.2±0.8)×104个和(3.9×0.6)×104个.与对照组相比,调低EF-1 alpha表达显著降低了前列腺癌细胞株DU145迁移和侵袭能力到37.1%(t=13.9,P<0.05). 结论调低EF-1 alpha表达水平对前列腺癌细胞的迁移和侵袭能力有负面影响.EF-1 alpha基因在前列腺痛细胞局部侵袭中有重要作用.     

Characterization of one newly established esophageal cancer cell line CSEC from a high-incidence area in China

SUMMARY.  The Chaoshan littoral is located in a high-incidence area of esophageal cancer in the south of China. In this study, a new esophageal cancer cell line CSEC was established from a 47-year-old female Chinese patient in this district. The biological characters of the cultured cells were investigated, including morphology, ultrastructure, growth kinetic features, tumorigenicity, expression of tumor-associated antigen and cytogenetic features. CSEC cell line grew continuously with a doubling time of 39.5 h and had been passaged over 80 times. The CSEC cells possessed features of squamous epithelial cells with cytokeratin indicated by immunohistochemical staining and tonofilaments and desmosomes revealed by electron microscopy. Tumorigenicity to severe combined immunodeficient mice was confirmed and the tumors developed revealed well-differentiated squamous cell carcinoma, similar to the origin tumor from which the cell line derived. The cytogenetic analysis demonstrated hypertetraploid karyotypes. Chromosome structure aberrations were common and complicated. Immunohistochemical staining showed that CSEC cells were infected with HPV and over-expressed p53. In summary, the CSEC cell line is a well-differentiated esophageal squamous cell carcinoma cell line from a high-incidence area in southern China. It may provide a useful model for the pathogenesis and therapeutic research of esophageal squamous cell carcinoma.     

趋化因子配体16对人胰腺癌细胞株PANC1生物学行为的影响

目的探讨趋化因子配体16（CXCL16）对人胰腺癌细胞PANC1生物学行为的影响。方法取对数生长期的人胰腺癌细胞PANC1,分别加入50、1013、200ng／ml的重组人CXCL16（rhCXCL16）和抗CXCL16抗体处理4h,以不加rhCLCL16作为对照。采用MTT法检测细胞增殖,采用Mqrtigel基质检测细胞的黏附率,采用Transwell小室检测细胞侵袭和迁移能力。结果100ng／ml rhCXCL16处理PANC1细胞后,细胞增殖、对基质的黏附率、侵袭和迁移能力分别为0．264±0．021、（91．4±8．6）％、1．246±0．216、1．361±0．276,其中细胞对基质的黏附率、侵袭和迁移能力均显著高于对照组的（20．6±3．2）％、0．259±0．013、0．199±0．008（P〈0．01）,对细胞的增殖不影响;2130ng/ml rhCXCL16处理后,细胞对基质的黏附率、侵袭和迁移能力进一步提高到（92．1±6．3）％、1．511±0．174、1．600±0．208（P〈0．05）。结论CXCL16可诱导人胰腺癌细胞PANC1增强对基质的黏附性、侵袭性和迁移性。